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- A fast and efficient translational control system for conditional expression of yeast genes (2009)
- A new artificial regulatory system for essential genes in yeast is described. It prevents translation of target mRNAs upon tetracycline (tc) binding to aptamers introduced into their 5'UTRs. Exploiting direct RNA–ligand interaction renders auxiliary protein factors unnecessary. Therefore, our approach is strain independent and not susceptible to interferences by heterologous expressed regulatory proteins. We use a simple PCR-based strategy, which allows easy tagging of any target gene and the level of gene expression can be adjusted due to various tc aptamer-regulated promoters. As proof of concept, five differently expressed genes were targeted, two of which could not be regulated previously. In all cases, adding tc completely prevented growth and, as shown for Nop14p, rapidly abolished de novo protein synthesis providing a powerful tool for conditional regulation of yeast gene expression.
- Antibiotika aus Käse und Humus : eine Alternative bei Resistenzen - die Lantibiotika Nisin und Subtilin (2003)
- Krankheitserreger lernen schnell und gründlich. Manche von ihnen besiegen bereits alle therapeutisch zur Verfügung stehenden Antibiotika. Doch Biochemiker und Mikrobiologen haben die Suche nach Substanzen mit neuartigen Wirkmechanismen gestartet und sind in Milch und Erde fündig geworden.
- Correction : mapping of complete set of ribose and base modifications of yeast rRNA by RP-HPLC and mung bean nuclease assay (2017)
- In Table 1, there are missing column headers. Please see the complete, correct Table 1 here. There are several errors in Table 2. Please see the correct Table 2 here.
- Identification of a new ribose methylation in the 18S rRNA of S. cerevisiae (2015)
- Methylation of ribose sugars at the 2′-OH group is one of the major chemical modifications in rRNA, and is catalyzed by snoRNA directed C/D box snoRNPs. Previous biochemical and computational analyses of the C/D box snoRNAs have identified and mapped a large number of 2′-OH ribose methylations in rRNAs. In the present study, we systematically analyzed ribose methylations of 18S rRNA in Saccharomyces cerevisiae, using mung bean nuclease protection assay and RP-HPLC. Unexpectedly, we identified a hitherto unknown ribose methylation at position G562 in the helix 18 of 5′ central domain of yeast 18S rRNA. Furthermore, we identified snR40 as being responsible to guide snoRNP complex to catalyze G562 ribose methylation, which makes it only second snoRNA known so far to target three ribose methylation sites: Gm562, Gm1271 in 18S rRNA, and Um898 in 25S rRNA. Our sequence and mutational analysis of snR40 revealed that snR40 uses the same D′ box and methylation guide sequence for both Gm562 and Gm1271 methylation. With the identification of Gm562 and its corresponding snoRNA, complete set of ribose methylations of 18S rRNA and their corresponding snoRNAs have finally been established opening great prospects to understand the physiological function of these modifications.
- Identification of a novel methyltransferase, Bmt2, responsible for the N-1-methyl-adenosine base modification of 25S rRNA in "Saccharomyces cerevisiae" (2013)
- The 25S rRNA of yeast contains several base modifications in the functionally important regions. The enzymes responsible for most of these base modifications remained unknown. Recently, we identified Rrp8 as a methyltransferase involved in m1A645 modification of 25S rRNA. Here, we discovered a previously uncharacterized gene YBR141C to be responsible for second m1A2142 modification of helix 65 of 25S rRNA. The gene was identified by reversed phase–HPLC screening of all deletion mutants of putative RNA methyltransferase and was confirmed by gene complementation and phenotypic characterization. Because of the function of its encoded protein, YBR141C was named BMT2 (base methyltransferase of 25S RNA). Helix 65 belongs to domain IV, which accounts for most of the intersubunit surface of the large subunit. The 3D structure prediction of Bmt2 supported it to be an Ado Met methyltransferase belonging to Rossmann fold superfamily. In addition, we demonstrated that the substitution of G180R in the S-adenosyl-l-methionine–binding motif drastically reduces the catalytic function of the protein in vivo. Furthermore, we analysed the significance of m1A2142 modification in ribosome synthesis and translation. Intriguingly, the loss of m1A2142 modification confers anisomycin and peroxide sensitivity to the cells. Our results underline the importance of RNA modifications in cellular physiology.
- Identification of novel methyltransferases, Bmt5 and Bmt6, responsible for the m3U methylations of 25S rRNA in Saccharomyces cerevisiae (2013)
- RNA contains various chemical modifications that expand its otherwise limited repertoire to mediate complex processes like translation and gene regulation. 25S rRNA of the large subunit of ribosome contains eight base methylations. Except for the methylation of uridine residues, methyltransferases for all other known base methylations have been recently identified. Here we report the identification of BMT5 (YIL096C) and BMT6 (YLR063W), two previously uncharacterized genes, to be responsible for m3U2634 and m3U2843 methylation of the 25S rRNA, respectively. These genes were identified by RP-HPLC screening of all deletion mutants of putative RNA methyltransferases and were confirmed by gene complementation and phenotypic characterization. Both proteins belong to Rossmann-fold-like methyltransferases and the point mutations in the S-adenosyl-L-methionine binding pocket abolish the methylation reaction. Bmt5 localizes in the nucleolus, whereas Bmt6 is localized predominantly in the cytoplasm. Furthermore, we showed that 25S rRNA of yeast does not contain any m5U residues as previously predicted. With Bmt5 and Bmt6, all base methyltransferases of the 25S rRNA have been identified. This will facilitate the analyses of the significance of these modifications in ribosome function and cellular physiology.
- Mapping of complete set of ribose and base modifications of yeast rRNA by RP-HPLC and mung bean nuclease assay (2016)
- Ribosomes are large ribonucleoprotein complexes that are fundamental for protein synthesis. Ribosomes are ribozymes because their catalytic functions such as peptidyl transferase and peptidyl-tRNA hydrolysis depend on the rRNA. rRNA is a heterogeneous biopolymer comprising of at least 112 chemically modified residues that are believed to expand its topological potential. In the present study, we established a comprehensive modification profile of Saccharomyces cerevisiae’s 18S and 25S rRNA using a high resolution Reversed-Phase High Performance Liquid Chromatography (RP-HPLC). A combination of mung bean nuclease assay, rDNA point mutants and snoRNA deletions allowed us to systematically map all ribose and base modifications on both rRNAs to a single nucleotide resolution. We also calculated approximate molar levels for each modification using their UV (254nm) molar response factors, showing sub-stoichiometric amount of modifications at certain residues. The chemical nature, their precise location and identification of partial modification will facilitate understanding the precise role of these chemical modifications, and provide further evidence for ribosome heterogeneity in eukaryotes.
- Minimum information about a biosynthetic gene cluster (2015)
- A wide variety of enzymatic pathways that produce specialized metabolites in bacteria, fungi and plants are known to be encoded in biosynthetic gene clusters. Information about these clusters, pathways and metabolites is currently dispersed throughout the literature, making it difficult to exploit. To facilitate consistent and systematic deposition and retrieval of data on biosynthetic gene clusters, we propose the Minimum Information about a Biosynthetic Gene cluster (MIBiG) data standard.
- Partial methylation at Am100 in 18S rRNA of baker's yeast reveals ribosome heterogeneity on the level of eukaryotic rRNA modification (2014)
- Ribosome heterogeneity is of increasing biological significance and several examples have been described for multicellular and single cells organisms. In here we show for the first time a variation in ribose methylation within the 18S rRNA of Saccharomyces cerevisiae. Using RNA-cleaving DNAzymes, we could specifically demonstrate that a significant amount of S. cerevisiae ribosomes are not methylated at 2′-O-ribose of A100 residue in the 18S rRNA. Furthermore, using LC-UV-MS/MS of a respective 18S rRNA fragment, we could not only corroborate the partial methylation at A100, but could also quantify the methylated versus non-methylated A100 residue. Here, we exhibit that only 68% of A100 in the 18S rRNA of S.cerevisiae are methylated at 2′-O ribose sugar. Polysomes also contain a similar heterogeneity for methylated Am100, which shows that 40S ribosome subunits with and without Am100 participate in translation. Introduction of a multicopy plasmid containing the corresponding methylation guide snoRNA gene SNR51 led to an increased A100 methylation, suggesting the cellular snR51 level to limit the extent of this modification. Partial rRNA modification demonstrates a new level of ribosome heterogeneity in eukaryotic cells that might have substantial impact on regulation and fine-tuning of the translation process.
- Specialized box C/D snoRNPs act as antisense guides to target RNA base acetylation (2017)
- Box C/D snoRNAs are known to guide site-specific ribose methylation of ribosomal RNA. Here, we demonstrate a novel and unexpected role for box C/D snoRNAs in guiding 18S rRNA acetylation in yeast. Our results demonstrate, for the first time, that the acetylation of two cytosine residues in 18S rRNA catalyzed by Kre33 is guided by two orphan box C/D snoRNAs–snR4 and snR45 –not known to be involved in methylation in yeast. We identified Kre33 binding sites on these snoRNAs as well as on the 18S rRNA, and demonstrate that both snR4 and snR45 establish extended bipartite complementarity around the cytosines targeted for acetylation, similar to pseudouridylation pocket formation by the H/ACA snoRNPs. We show that base pairing between these snoRNAs and 18S rRNA requires the putative helicase activity of Kre33, which is also needed to aid early pre-rRNA processing. Compared to yeast, the number of orphan box C/D snoRNAs in higher eukaryotes is much larger and we hypothesize that several of these may be involved in base-modifications.
